Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/65—Raman scattering
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/47—Scattering, i.e. diffuse reflection
  • G01N2021/4704—Angular selective

Definitions

• the present invention relates generally to the evaluation of samples through use of Raman spectroscopy adapted to direct electromagnetic radiation to intersect the sample at a plurality of angles: and more particularly relates to such methods wherein the Raman scattered electromagnetic radiation from a plurality of angles of incidence is used to evaluate one or more properties of the sample under investigation.
• a source of electromagnetic radiation such as a laser
• the excitation energy will be scattered.
• Most of the light scattered by the sample will be scattered elastically; this light is at an unshifted wavelength and may be detected after leaving the specimen.
• a relatively small portion of the laser light is scattered inelastically as a result of coming into contact with the sample.
• This inelastically scattered light exits the specimen at shifted wavelengths which are at both higher and lower energy states than the original laser wavelength.
• the light shifted to longer wavelengths is called the Stokes-shifted Raman signal, and the light shifted to shorter wavelengths is called the anti-Stokes Raman signal.
• the amount of the shift reflects the vibrational spectrum of the sample under examination.
• This Raman shift spectrum may be detected and analyzed, such as through use of a spectrograph to evaluate one or more properties or characteristics of the sample under examination.
• a limitation of current Raman spectroscopy systems is a relatively limited capability to interact with a sample at different depths into the sample. Systems such as confocal Raman probes have been used to provide some variability of depth of investigation, However, these systems are relatively inefficient; and in view of the relatively small amount of Raman scattering signal that is typically available, will not be well-suited to some applications.
• the methods and apparatus described herein provide a novel methodology for variable angle Raman spectroscopy, in which an excitation beam of electromagnetic radiation will be caused to intersect the sample under investigation at a plurality of angles of incidence, so as to provide Raman scattering spectra at each angle.
• the excitation beam will be a single wavelength.
• multiple excitation beams, each at a single wavelength may be used to excite the sample to induce the Raman scattering.
• One example use of measuring such spectra at multiple angles of incidence is to enable evaluation at a plurality of depths within the sample.
• the range of the plurality of angles of incidence utilized will be very near the critical angle of incidence; and thus, such methods may be termed "peri-critical Raman spectroscopy," In many examples as described herein, the range of the angles of incidence will include, and extend to either side of, the critical angle.
• Figure 1 depicts a flowchart depicting an example method for variable angle Raman spectroscopy as further described herein.
• Figure 2 depicts a block diagram representation of a variable angle Raman spectroscopy system as may be used to perform the method of Figure 1.
• Figure 3 depicts a block diagram representation of a Raman detector as may be used in the system of Figure 2.
• Figure 4 depicts a flowchart of example functionality of the controller assembly in the system of Figure 2.
• Figure 5 depicts an alternative configuration for an electromagnetic beam directing assembly.
• Figure 6 depicts a block diagram representation of an example of a controller architecture suitable for use in the system of Figure 2.
• references to “one embodiment” or “an embodiment,” or to “one example” or “an example” mean that the feature being referred to is, or may be, included in at least one embodiment or example of the invention.
• references to “an embodiment” or “one embodiment” or to “one example” or “an example” in this description are not intended to necessarily refer to the same embodiment or example; however, neither are such embodiments mutually exclusive, unless so stated or as will be readily apparent to those of ordinary skill in the art ha ving the benefit of this disclosure.
• the present invention can include a variety of combinations and/or integrations of the embodiments and examples described herein, as well as further embodiments and examples as defined within the scope of all claims based on this disclosure, as well as all legal equivalents of such claims.
• a "processor-based system' ' ' or “processing system” as used herein includes a system using one or more microprocessors, microcontrollers and/or digital signal processors or other devices having the capability of running a "program,” (all such devices being referred to herein as a “processor”).
• a “program” is any set of executable machine code instructions, and as used herein, includes user-level applications as well as system-directed applications or daemons.
• FIG. 1 depicts a flow chart of an example methodology 100 for peri-critical Raman spectroscopy.
• electromagnetic radiation at a single wavelength, will be directed to transmit through a prism to engage a sample, as indicated at block 102.
• the sample will be discussed as a tissue sample, as that is one of many applications in which the described method may advantageously be used.
• the path of the electromagnetic beam will be directed, as identified at block 104, such that it intersects the tissue sample at a plurality of angles within a selected range of angles.
• a primary range of interest will be when the beam is directed at angles of incidence very close to, and including, the critical angle; for example, within a range beginning 0.20 degree above the critical angle or less, such as beginning at 0.10 degree above the critical angle. And in many applications the range will extend through the critical angle of incidence, and for a similar range below the critical angle.
• the critical angle of incidence for a sample has been discussed in my co-pending, published PCT application No.
• n ⁇ represents the refractive index of the prism
• m represents the refractive index of the sample.
• the manipulation of the beam to cause the described intersection at a plurality of precisely controlled angles can be accomplished through a variety of mechanisms.
• the beam directing mechanism can be relatively direct, such as through use of a precision drive to incrementally rotate (or otherwise change) the position of the source of the electromagnetic beam, which will most commonly be a laser, through a range of positions, such as through a plurality of rotational positions relative to the described prism and tissue sample.
• beam directing mechanisms may be implemented, such as, for example, directing the electromagnetic beam from the laser (or other source) to die prism through use of one or more mirrors or intermediate deflection prisms, and by moving the mirror(s) (or deflection prisms) through a range of positions relative to the fixed source and fixed prism contacting the sample.
• each detected Raman scattering spectrum will preferably be correlated with the angle of incidence resulting in that spectrum.
• This detected Raman scattering will typically provide a substantially complete spectrum, at least within a range of interest. As will be understood by those skilled in the art, in many applications it will be desirable to remove detected radiation which is at the frequency of the initial excitation wave, as such does not represent Raman scattered electromagnetic radiation.
• the described methods and apparatus for peri-critical Raman spectroscopy described herein are particularly suited to providing vibrational spectroscopy information, thereby reflecting the molecular structure of the sample under consideration.
• the useful information is obtained in up to 4000 wave numbers of shift.
• a wavelength of 1 ⁇ represents 10,000 wave numbers, and the wavelengths of value are from 1 to 1 ,6 ⁇ , as those light wavelengths will define the 4000 wave number span of useful vibrational spectral information.
• the detected Raman scattering may be used to evaluate one or more properties or parameters of the sample under investigation.
• the exact manner by which this evaluation is performed may be at least partially through any of a number of techniques that are known to those skilled in the art.
• the detected scattered electromagnetic spectra may be compared to one or more reference spectra.
• These reference spectra may be used to determine the presence or absence of one or more properties, such as variations in the molecular composition of the sample under investigation, in addition to comparing one or more the individual spectra to a reference, all or some portion of the spectnim obtained throughout the range of the angle of excitation may be combined to identify depths within the sample where the data reflects a change in the molecular composition of the sample under investigation.
• tissue such as human or other mammalian tissue
• a natural example would be examination of the skin.
• One example application would be to examine the skin for spectral patterns which are consistent with spectral patterns observed where a melanoma or similar abnormality is present in the skin.
• Figure 1 also depicts a pair of example additional steps which may optionally be performed in the example method.
• block 1 10 describes an operation that may be performed to approximately identify the critical angle, and from that to determine a range of angles to be used in the operation defined at block 104.
• the electromagnetic beam angle of intersection might be incrementally scanned through a relatively broad range expected to contain the critical angle, for example a range on the order of 4 or 5 degrees; and the incremental positioning might be done at a relatively coarse resolution in order to minimize the time required for scanning across the entire range. For example, a resolution of 0.1 degrees to 0.2 degrees might be used beneficially for some applications.
• the critical angle may be determined within a relatively narrow range.
• the scanning operation of block 104 may be performed over a much more limited range, for example only about 0.10 degrees to either side of the critical angle.
• the resolution of the incremental positioning of each scan across that range may be much smaller. For example, positioning within increments of a fraction of a millidegree is possible with existing hardware, in actual practice, the preferred incremental range may be often within the range of .0003 degrees up to 0.01 to 0.02 degrees, for example. In general, deeper depth of sampling is attained by longer wavelength excitation laser light, but with such longer wavelengths, greater angular resolution will be required around the critical angle.
• the wavelength used and the resolution requirements can be variable in response to the sample being interrogated and/or the needed depth of investigation.
• the coarse scanning operation of block 110 in some systems which are constructed to include a detector for reflected electromagnetic radiation, it will be possible to use just the reflectance detector to identify the critical angle. Due to the significant difference in the reflected signal at the critical angle, this may allow the coarse scan to be performed, and the data analyzed, relatively efficiently, as compared to collecting all the data of the Raman scattered spectra at each incremental position.
• the detected radiation from either Raman scattering or reflection, is processed to correlate the angle of incidence versus the detected intensity of radiation, to determine the inflection point that will identify the critical angle.
• peri-critical spectroscopy has been used in absorption spectroscopy, such as attenuated total reflectance (ATR) spectroscopy.
• absorption spectroscopy such as attenuated total reflectance (ATR) spectroscopy.
• ATR attenuated total reflectance
• Raman spectroscopy can provide a specific quantitative measurement, such as the degree of presence of a specific constituent in the sample, where the constituent is one that absorbs light at the excitation wavelength.
• Raman spectroscopy as described herein provides an essentially full-spectrum analysis, and thus is better suited to applying a relatively qualitative characterization as to one or more parameters of the sample under investigation.
• each obtained spectrum can be representative of a respective depth in the sample, the depth being a function of the angle of incidence and the properties of the resulting evanescent wave.
• the Raman-shifted spectral data detected for each incremental angle of incidence can be correlated to provide a three dimensional representation of the sample, wherein a change in the spectral information from one spectrum to another one reflects some change in the molecular composition between the two depths which correlate to the spectra in question.
• the spectra of interest may be individually compared with one or more references to identify the sample composition causing the identified change. Additionally, the spectra may be correlated with one another, either in their entirety, or within certain wavelengths of interest to generate a correlated spectrum signal which may be compared to one or more references.
• Another advantage of the described methodology is an improvement in the relative volume of Raman scattered light.
• a laser beam interacts with tissue, significant scattering can occur.
• internal reflection spectroscopy is relatively immune to the effects of such scattering in tissue; and therefore a greater portion of the returned light is the Raman inelastic scatter. This greater portion of returned Raman scatter allows greater resolution between the Raman scatter and the non-Raman scatter.
• Raman spectroscopy system 200 depicted in an operative relationship with a sample to be evaluated.
• sample 202 will again be described as tissue sample, such as human or other mammal tissue.
• System 200 includes an illumination source, such as for most applications a laser assembly 204.
• Laser assembly 204 may optionally include an interferometer or other mechanism to assist in coliimating the beam from laser 204.
• Laser assembly 204 will be mounted on an appropriate mechanism for incrementally moving laser 2042 change the angle of incidence of the laser beam relative to tissue sample 102.
• An example of one suitable precision drive for incremental control of positioning of laser 204 is the model RGV100 from Newport Corporation of Irvine, California. This precision rotational drive is capable of unidirectional repeatability of 0,3 milli degrees.
• a single diode laser will often be desirable.
• Such diode lasers can provide a beam of sufficient power for examination of such tissue and can be relatively portable, as may be desired for some applications of the described systems,
• a significant constraint that will be imposed on the system is the power of the electromagnetic radiation that can be used to excite the sample.
• the mid-infrared range will be considered to refer to wavelengths in the range of 2,5 to 20 microns; and the near-infrared range will be considered to refer to wavelengths in the range of approximately 700 nm to 2.5 microns.
• the described techniques and systems are used to evaluate tissue samples as described in the examples herein, it will often be appropriate to use wa velengths within the range of approximately 700 nm to 1 micron.
• the electromagnetic beam is directed through a prism 208 that will have a sample-engaging surface be placed in contact with the sample 202.
• prism 208 A number of possible configurations for prism 208 may be contemplated. As just one example, a relatively straightforward configuration will be for the prism to have a cross section in the form of a truncated triangle thereby forming a symmetrical trapezoidal cross section.
• the sample-engaging surface 220 extends in parallel relation to a spectra-emitting surface 222, through which the Raman scattered radiation will pass.
• This configuration for prism 208 will allow the Raman scattered radiation to pass from a spectrally emitting sample through the prism 208 to a detector 210 essentially directly opposite the sample. Additionally, reflected Sight will transmit along axis 218 to reflection detector 216. Reflection detector 216 is an optional component for system 200, which is indicated by the dashed lines identifying the detector and its connections within the system.
• the choice of the material for the prism may vary depending upon the nature of the samples to be examined. For many applications, it will be preferable to have a prism with a refractive index that is higher than that of the sample but only by a relatively small amount. In the case of spectroscopy systems intended for evaluating living human tissue, such tissue will typically have a refractive index falling within a range of approximately 1 .33 to 1 .5. If we were to assume a 1 ⁇ wavelength for the excitation wave, then barium fluoride or zinc sulfide would typically be appropriate choices.
• Raman scatter detector 210 is described in more detail in reference to Figure 3, below.
• Raman scatter detector to 10 is essentially a spectrograph configured to detect radiation within the frequency range of interest for the sample and excitation frequency utilized. While a single element detector may be used in some applications, the use of a multiple pixel array detector will be desirable for many applications.
• controller assembly 212 is not necessarily a single device or component, but may be embodied in multiple components that together provide the necessary functionality. In the case where controller assembly is formed a separate components, it should be clearly understood that these components may not be located in one general location, but may be distributed, and connected as needed, for example through one or more networks, including local area networks (LAN), wide area networks (WAN) and/or the Internet. Some or all of the described control functionality may be performed through one or more application programs functioning on a general purpose computer.
• LAN local area networks
• WAN wide area networks
• Internet Internet
• the controller assembly 212 may be a dedicated purpose device, or may include a dedicated purpose device, specifically adapted to provide the necessary functionality.
• the depicted output devices 214 may in fact be a portion of the control ler assembly 212, or may be separate components tha t are not necessarily part of the system at all.
• the output devices might be remote terminals, printers or databases as may be appropriate to facilitate further analysis, use or maintaining of the acquired data and/or analysis results.
• most embodiments of a controller assembly 212 will include one or more processors, with each processor executing a plurality of instructions that are collectively retained in one or more instances of machine-readable storage medium.
• controller assembly 212 In most instances these machine-readable storage media will be found within controller assembly 212, however such placement is not mandator ⁇ ' .
• machine-readable instructions might be retained in a machine-readable medium remote from the controller but communicated to the controller assembly 212 across a wired or wireless network.
• detector 210 which may be a generally conventional spectrograph.
• Detector 210 will preferably include a relatively high efficiency imaging device 302 so as to capture the relatively small signals typically represented by Raman scattering.
• a high efficiency CCD or CMOS imager is useful.
• detector 210 will include a lens 304 to capture the elastically scattered light coming from prism 208.
• the IXON X3 EMCCD camera available from Andor Technology of Harbor, Northern Ireland, is suitable for many applications.
• detector 210 will preferably include a diffraction grating 308.
• the diffraction grating will split and diffract the impinging light into separate beams.
• An example of an appropriate grating would be a 1200 line per millimeter grating; which preferably would be a holographic grating.
• an appropriate edge filter may be used, wherein the edge filter would either filter the excitation wavelength and all wavelengths below it, or alternatively would filter out the excitation wavelength and all wavelengths about it.
• controller assembly 212 depicts a flow chart for basic operations to be performed under control of controller assembly 212.
• controller functionality 400 of Figure 4 is to orient the laser (or other beam directing mechanism) such that the scan will start at the desired angle of incidence.
• the start angle of incidence may, in some cases be pre-programmed, or in other cases may be input by a user.
• Controller assembly 212 will send appropriate signals to orientation device 206 to achieve the desired placement.
• controller assembly 212 track the position of the mechanism and/or monitor the directing mechanism for indication of the position, in order to allow correlation of spectral measurements with the angle of incidence that resulted in each detected spectrum.
• the laser will be activated for a given time period.
• the given time may be a selected interval for discrete measurement.
• the laser may be left on continually, as it is scanned through the incremental positions of the selected range.
• Controller assembly 212 will then receive the data representative of detected scattering from detector 210. It wi ll be apparent to those skilled in the art having the benefit of this disclosure that the timing of the receiving of this information by the controller assembly will, at least in most embodiments, not be critical, and the data might be retained in detector 210 for some period of time before being transmitted to controller assembly 212,
• the data representative of the detected scattering will typically, and often preferably, include the raw detected spectra] data.
• the data representative of the detected scattering may be, or may include, processed spectral data.
• a reflection detector 216 is included in example system 200 as discussed earlier herein, then reflected light received by detector 216 may also be received, as identified at block 408. Subsequently, the received spectral information from Raman detector 210 and/or from reflection detector 216 will be recorded in some manner. In most embodiments, the recording will include a digital representation of the received information being stored in a machine- readable medium either in or in communication with controller assembly 212.
• controller assembly 212 will make a determination as to whether the preceding scan position was the end of the range of the scan (ie, whether the last position was the last position within a scan of multiple positions). If the answer is no, controller assembly 212 will increment the position of the laser to the next position within the scan range (at block 414) and the process will return to block 404 to again activate the laser (unless the laser is continuously activated during the scan). Once the system finally determines at block 412 that the preceding scan position was the final position within the scan, then the flow proceeds to block 416 where controller assembly 212 will, in at least some embodiments, perform some degree of processing of the data, as discussed earlier herein. Subsequently, the data and/or the results of the processing will be output to appropriate devices.
• the example flowchart depicts the processing being done at the end of the data collection, there are circumstances where it may be desirable to do some types of processing during the data collection.
• the peri-critical Raman spectroscopy system includes a reflection detector, as indicated at 216 in Figure 2
• signals from that detector may be used to monitor the stability of the excitation laser. In some examples, that monitoring would preferably be performed during the scanning of the excitation laser through the selected range.
• signals from the reflection detector could be used to normalize the intensity of the received Raman scatter spectrum during processing of the scattering data.
• FIG. 5 that figure depicts a block diagram representation of an alternative optical assembly 500 that could be used in a peri- critical Raman spectroscopy system otherwise substantially in the form of that described in reference to Figure 2.
• laser 502 is in a fixed position, and is oriented such that the electromagnetic beam may be reflected by a movable mirror (or an intermediate deflection prism) 504 to transmit through prism 208 to tissue sample 202
• Movable mirror (or deflection prism) 504 may be mounted on the same form of an precision rotational controller 506 as described in reference to Figure 2 for use with the laser in that embodiment.
• multiple mirrors or deflection prism may be utilized in place of the single assembly depicted in Figure 5.
• the deflection assemblies are only depicted relative to the electromagnetic beam input, of course radiation returning from the sample through the prism may also be directed to one or more desired paths toward the one or more detectors through use of similar mirrors or deflection prisms.
• all other basic operation of the optical system remains the same.
• a first wavelength may interact with the sample to provide a first range of penetration into the sample; and a second wavelength may be selected to interact with the sample at either a shallower or deeper depth throughout the contemplated range of angles of incidence.
• the use of multiple wavelengths may be useful in avoiding barriers to effective Raman spectroscopy, For example, with Raman spectroscopy of some types of samples, excitation at certain wavelengths can cause fluorescence, which interferes with effective measurement of Raman scattering. By having multiple wavelengths of excitation, if a first set of scattering data resulting from a first wavelength is less than optimal, such as due to the described fluorescence, the one or more alternative wavelengths may be used to provide satisfactory measurements.
• controller 600 depicts block diagram representation of an example architecture for controller 600, such as may be used to provide some or all of the functions of a controller assembly, for which one example was described in reference to Figure 4.
• controller assembly 212 would include one or more microprocessors which will operate pursuant to one or more sets of instructions for causing the machine to perform any one or more of the methodologies discussed herein.
• the example controller assembly 600 includes a processor 602 (e.g., a central processing unit (CPU) a graphics processing unit (GPU) or both), a main memory 604 and a static memory 606, which communicate with each other via a bus 608.
• the controller assembly 600 may further include a video display unit 610 (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)).
• the controller assembly 600 also includes an alphanumeric input device 612 (e.g., a keyboard, mechanical or virtual), a cursor control device 614 (e.g., a mouse or track pad), a disk drive unit 616, a signal generation device 618 (e.g., a speaker) and a network interface device 620,
• the disk drive unit 616 includes a machine-readable medium 622 on which is stored one or more sets of executable instructions (e.g., software 624) embodying any one or more of the methodologies or functions described herein.
• a solid-state storage de v ice such as those comprising flash memory may be utilized.
• the software 624 may also reside, completely or at least partially, within the main memory 604 and/or within the processor 602 during execution thereof by the controller assembly 600, the main memory 604 and the processor 602 also constituting machine -readable media.
• the instructions may be only temporarily stored on a machine- readable medium within controller 600, and until such time may be received over a network 626 via the network interface device 620.
• machine-readable medium 622 is shown in an example embodiment to be a single medium, the term “machine-readable medium” as used herein should be taken to include a single medium or multiple media (e.g., a centralized or distributed database, and/or associated caches and servers) that store the one or more sets of instructions.
• the term “machine-readable medium” or “computer-readable medium” shall be taken to include any tangible non- transitory medium which is capable of storing or encoding a sequence of instructions for execution by the machine and that cause the machine to perform any one of the methodologies.

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